Compositions and methods for improved protein production

ABSTRACT

The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG.  1 , and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

CLAIM OF PRIORITY

This application claims priority to provisional application 60/840,750filed on Aug. 29, 2006, the contents of which are hereby incorporated byreference in their entireties.

GOVERNMENT SUPPORT

Portions of this work were funded by Subcontract No. ZCO-30017-01 withthe National Renewable Energy Laboratory under Prime Contract No.DE-AC36-99GO10337 with the U.S. Department of Energy. Accordingly, theUnited States Government may have certain rights in this invention.

FIELD

The invention relates to novel host cells with improved proteinproduction, methods of producing such host cells and uses thereof.

INTRODUCTION

Enzyme washing is commonly used as a wet process technique to improvetextile handling, appearance and other surface characteristics of, e.g.,cottons and cotton blends in the industry. One example of the successfulapplication of enzyme technology in the textile industry is thereplacement of traditional stone washing (which is very time consumingand labor intensive) in denim processing by cellulase washing.Hydrolysis of cellulase, a major component of cotton, with cellulase isuseful for the biopolishing of cotton fabrics, which enhances theiraesthetic performance by cleavage of glycosidic bonds in cellulosemolecules. Cellulases are important industrial enzymes used, forexample, in the processing of textiles and in detergents. Cellulases areenzymes that hydrolyze cellulose (e.g., α-1,4-D-glucan linkages) andproduce as primary products glucose, cellobiose andcellooligosaccharides. The cellulases used in the textile industry areproduced by several different microorganisms and comprise severaldifferent enzyme classifications including those identified asexo-cellobiohydrolases (CBH), endoglucanases (EG) and β-glucosidases(BG) (M. Schulein, Methods in Enzymology, vol. 160, pp. 235-242 (1988)).

Therefore, cellulases, and components thereof, either individually or incombination, are useful in treating textiles. Additional benefits tousing cellulases to treat cotton-containing fabrics include the removalof sizing from the fabric (sizing is a composition used to stiffenfabric so it is easier to handle in the manufacture of, for example,garments) removing fuzz and pills from the surface of the fabric andgiving a stone-washed appearance and feel to the fabric. Still,improvements in the effectiveness and efficiency of cellulase treatmentof fabrics will be beneficial to the garment and textile industries aswell as other industries such as in the manufacture of detergents and inthe manufacturing of fuel ethanol from biomass.

Despite intensive research related to the use of cellulases inindustrial processes, cellulases known and used in the art have shownsignificant drawbacks. For example, many cellulases have beenproblematic due to low activity, poor alkaline or acid stability, poortemperature stability and poor oxidative stability. More importantly,cellulase production by microorganisms is often low and, therefore,inefficient from a commercial standpoint. Therefore, what is needed arenew strains of microorganisms and new nucleotide sequences that improvethe efficiency of cellulase production.

SUMMARY

The applicants have discovered that disruption of specific nucleotidesequences in a host cell results in improved production or a desiredprotein by such modified host cells. The applicants have also identifiedmolecular basis responsible for the improved protein production.Accordingly, the invention features novel host cells suitable for theenhanced production of a desired polypeptide compared to the parentstrain, methods of producing a desired polypeptide from the said hostcells and the specific disrupted nucleotide sequences (SEQ ID NOS.: 1-6)responsible for the improvements in production of a desired polypeptide.

In a first embodiment there is provided a modified host cell. Themodified host cell may be a fungi or a bacterium. The modified host cellcomprises deletion or disruption of specific nucleotide sequences thatresults in the improved expression and/or secretion of a desiredpolypeptide. The specific nucleotide sequence that may be disrupted isselected from 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1,and are SEQ ID NOS.: 1-6, respectively, or sequences having 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ IDNOs:1-6, or sequences that have been codon optimized for the specifichost cell. In one aspect the fungi is a filamentous fungi. In a furtheraspect, the filamentous fungi is selected from Trichoderma, e.g.,Trichoderma reesei, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicolainsolens; Chrysosporium sp., including C. lucknowense; Gliocladium sp.;Aspergillus sp.; Fusarium sp., Neurospora sp., Hypocrea sp., andEmericella sp. In another aspect there is provided a host cell that hashad the endogenous genes disrupted or genes corresponding to thenucleotides provided for herein provides improved protein productionover the parent host cell strain (i.e., an unmodified host cell).

In one embodiment there is provided a host cell having a mutation ordeletion of part or all of a gene having the sequence selected from atleast one sequence set forth in any one of SEQ ID NOs:1-6, and saidmutation or deletion results in the enhanced production of a desiredpolypeptide compared to the parent host cell.

In an aspect, the host cell is a filamentous fungus. In another aspect,the desired protein may be a homologous or heterologous to the hostcell. In a further aspect the heterologous proteins may be selected fromthe group consisting of hormones, enzymes, growth factors, andcytokines. In a yet further aspect, the enzyme is selected from thegroup consisting of proteases, carbohydrases, lipases, isomerases,racemases, epimerases, tautomerases, mutases, transferases, kinases andphosphatases.

In a second embodiment there is provided a method for the production ofa heterologous protein in a transformed filamentous fungus host cellcomprising the steps of:

-   -   (a) obtaining a filamentous fungus host cell comprising a        nucleic acid encoding said heterologous protein wherein said        host cell contains a mutation or deletion in at least one        nucleic acid sequence having the sequence set forth in any one        of SEQ ID NOs:1-6, wherein said mutation or deletion results in        the enhanced production of the heterologous protein compared to        a parent filamentous fungus: and    -   (b) growing said filamentous fungus host cell under conditions        suitable for the expression of said heterologous protein.

In certain aspects the nucleic acid that is mutated or deleted is atleast SEQ ID NO:1 or SEQ ID NO:2. In a third embodiment there isprovided an isolated nucleotide sequence selected from a groupconsisting of SEQ ID NOs: 1-6. In one aspect the nucleotide sequence hasbeen modified. The modification may be selected from truncation,deletion, mutation or other means of inactivation. Also provided hereinare vectors comprising at least one isolated nucleotide sequenceselected from a group consisting of SEQ ID NOs: 1-6 wherein thenucleotide sequence has been modified.

In a fourth embodiment there is provided a method of producing amodified host cell said method comprising:

-   -   (a) obtaining a parental host cell strain    -   (b) transforming said parental cell strain with the vector        comprising at least one isolated nucleotide sequence selected        from a group consisting of SEQ ID NOs: 1-6 wherein the        nucleotide sequence has been modified,    -   (c) selecting modified host cells        wherein said modified host cells produce more homologous protein        than the parental host cell.

In a fifth embodiment there is provided a method of producing aheterologous desired polypeptide said method comprising:

-   -   (a) obtaining a parental host cell strain    -   (b) transforming said parental cell strain with a vector        encoding a desired polypeptide;    -   (c) transforming said parental cell strain with a vector        comprising at least one isolated nucleotide sequence selected        from a group consisting of SEQ ID NOs: 1-6 wherein the        nucleotide sequence has been modified to produce a modified host        cell    -   (d) selecting modified host cells that produce said heterologous        desired polypeptide        wherein steps (b) and (c) may be done in any order or        simultaneously. The suitable sterile growth medium additionally        comprises an inducer of cellulase production selected from one        or more of cellulose, lactose, sophorose and glucose/sophorose.        The method may additionally comprises the at least partial        purification of cellulases produced by said culture.

In a sixth embodiment there is provided a method for the producing anovel strain of T. reesei using insertional mutagenesis wherein saidnovel strain of T. reesei has superior total protein or cellulaseproduction as compared to the parent strain of T. reesei, comprising:

-   -   (a) preparing a population of competent Agrobacterium sp., cells        by electroporating into competent Agrobacterium sp., cells an        expression vector comprising, in operable condition, the left        and right T-DNA boarder regions, pV51 plasmid origins for        replication in Agrobacterium sp. and bacterial markers to confer        resistance to chloramphenicol to create a population comprising        transformed Agrobacterium sp., cells;    -   (b) selecting for Agrobacterium from said population of step        (a);    -   (c) inoculating a culture of T. reesei spores with the        Agrobacterium sp. transformants of step (b) to create an        induction culture;    -   (d) culturing said induction culture of step (c) at about 18° C.        and for about 24 hours to create a population comprising;    -   (e) transferring samples of said population of transformed T.        reesei of step (d) to selective medium and isolating colonies        of T. reesei effective in degrading cellulose; and    -   (f) comparing the effectiveness of cellulose degradation between        the T. reesei of the isolated colonies of step (e) and the        non-transformed parent strain, wherein said T. reesei of the        isolated colonies of step e are superior to in cellulose        degradation when compared to the non-transformed parent strain.        The Agrobacterium sp, cells are selected from Agrobacterium        tumefaciens and Agrobacterium rhizogenes.

DRAWINGS

FIG. 1, shows the nucleic acid sequences of SEQ ID NO.: 1 of T. reesei8k and SEQ ID NO.: 2 from T. reesei 7p and SEQ ID No: 3 from T. reesei7E, SEQ ID NO 4: from T. reesei 9G, SEQ. NO. 5 from T. reesei 8Q and SEQNO. 6 from T. reesei 203.

FIG. 2 (a) shows an expression vector used for the transfection of T-DNAborder regions into Agrobacterium.

FIG. 2 (b) shows the pyr4 disruption contained within the expressionvector of FIG. 2 (a).

FIG. 3 is a schematic for the pPZP100/pyr4 vector.

FIG. 4 shows a representation of spore growth in the Toyama screen.

DESCRIPTIONS OF VARIOUS EMBODIMENTS

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

A “polypeptide of interest”, “protein of interest”, “desiredpolypeptide” and “desired protein” are used interchangeably herein.

The terms “protein(s)” and “polypeptide(s)” are used interchangeablyherein. The conventional one-letter or three-letter code for amino acidresidues is used herein.

A “heterologous promoter,” as used herein is a promoter which is notnaturally associated with a gene, gene portion or a purified nucleicacid.

In the present context, the term “substantially pure polypeptide” meansa polypeptide preparation which contains at the most 10% by weight ofother polypeptide material with which it is natively associated (lowerpercentages of other polypeptide material are preferred, e.g. at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most ½% by weight). Thus, it ispreferred that the substantially pure polypeptide is at least 92% pure,i.e. that the polypeptide constitutes at least 92% by weight of thetotal polypeptide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polypeptidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polypeptides disclosed herein arein “essentially pure form”, i.e. that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyassociated. This can be accomplished, for example, by preparing thepolypeptide by means of well-known recombinant methods. Herein, the term“substantially pure polypeptide” is synonymous with the terms “isolatedpolypeptide” and “polypeptide in isolated form”.

A “purified preparation of cells,” as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells as a portion of the total number ofcells.

In the context of the present invention the term “biologically pure” isdefined as having substantially, e.g., the above mentioned T. reeseistrains as the only living organism in the culture or the predominantliving organism in the culture and that the culture is substantiallyfree of other living organisms. The culture need not be 100% free ofother organisms providing the other organisms do not substantiallyinterfere with the growth of the T. reesei strains of the presentinvention.

The ability to culture T. reesei for the production of cellulase enzymesis known in the art as is exemplified in the Examples section, infra,and in U.S. Pat. Nos. 4,797,361, 4,762,788 and 4,472,504.

A “substantially pure nucleic acid,” e.g., a substantially pure DNA,RNA, etc., is a nucleic acid which is one or both of: 1) not immediatelycontiguous with either one or both of the sequences, e.g., codingsequences, with which it is immediately contiguous (i.e., one at the 5′end and one at the 3′ end) in the naturally-occurring genome of theorganism from which the nucleic acid is derived; or 2) which issubstantially free of a nucleic acid sequence with which it occurs inthe organism from which the nucleic acid is derived. The term includes,for example, a recombinant DNA which is incorporated into a vector,e.g., into an autonomously replicating plasmid or virus, or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment) independent of other DNA sequences.

The term “heterologous” with reference to a polynucleotide or proteinrefers to a polynucleotide or protein that does not naturally occur in ahost cell. In some embodiments, the protein is a commercially importantindustrial protein. It is intended that the term encompass proteins thatare encoded by naturally occurring genes, mutated genes, and/orsynthetic genes. The term “homologous” with reference to apolynucleotide or protein refers to a polynucleotide or protein thatoccurs naturally in the host cell.

“Cellulase,” “cellulolytic enzymes” or “cellulase enzymes” meansbacterial, or fungal exoglucanases or exocellobiohydrolases, and/orendoglucanases, and/or β-glucosidases. These three different types ofcellulase enzymes act synergistically to convert cellulose and itsderivatives to glucose.

Many microbes make enzymes that hydrolyze cellulose, including the woodrotting fungus Trichoderma, the compost bacteria Thermomonospora,Bacillus, and Cellulomonas; Streptomyces; and the fungi Trichoderma,Humicola, Aspergillus and Fusarium. The enzymes made by these microbesare mixtures of proteins with three types of actions useful in theconversion of cellulose to glucose: endoglucanases (EG),cellobiohydrolases (CBH), and beta-glucosidase.

The term “reverse genetics,” as defined herein, refers to a strategy todetermine a particular gene's function by studying the phenotypes withalterations in the gene of interest. In other words, after obtaining thestrain with a desired phenotype, e.g., altered morphology, aninvestigation to determine the genetic change responsible for thephenotype is undertaken. Various techniques can be used for reversegenetics including the use of transposons and REMI(Restriction-enzyme-mediated integration). This differs from classicalwherein one tries to determine the phenotype resulting from geneticchange. To learn the influence a sequence has on phenotype, or todiscover its biological function, researchers can engineer a change ordisruption in the DNA. After this change has been made a researcher canlook for the effect of such alterations in the whole organism. In thepresent invention, insertional mutagenesis has been done by transformingin the pyr4 gene into T. reesei. In most cases, a single copy of the pyr4 gene integrates randomly into the T. reesei genome disrupting thegene(s) that is present at that site. Since the sequence of the pyr 4gene is know, it acts as a tag and can be used to detect any geneticchanges that has been made. In this case, pyr4 is also used as ahomologous selectable marker for the transformation of T. reesei (Smith,et al., Sequence of the cloned pyr4 gene of Trichoderma reesei and itsuse as a homologous selectable marker for transformation. Curr. Gen,19:27-33, 1991). Other methods can be used to create disruptions in DNAfor reverse genetics screens including random deletions, insertions andpoint mutations, directed deletions and point mutations, gene silencingand interference using transgenes.

The term “T-DNA” as defined herein, refers to sequences of DNA common toAgrobacterium that facilitates the transfer of DNA into plant genomes.T-DNA is used by molecular biologists to permit the transfer of selectedDNA into a plant genome for the purpose of, for example, creatinginsertional mutants for the purpose of performing reverse genetics. Innature, the T-DNA of Agrobacterium facilitates the transfer of DNA intoa plant host causing crown gall disease. For the purposes oftransformation, only the border regions of the T-DNA sequences are usedthereby permitting the insertion of desired DNA sequences. In thepresent invention, pyr4 genes were inserted into the T. reesei genomeusing T-DNA border sequences.

As used herein, “microorganism” refers to a bacterium, a fungus, avirus, a protozoan, and other microbes or microscopic organisms.

As used herein, “derivative” means a protein which is derived from aprecursor protein (e.g., the native protein) by addition of one or moreamino acids to either or both the C- and N-terminal end, substitution ofone or more amino acids at one or a number of different sites in theamino acid sequence, deletion of one or more amino acids at either orboth ends of the protein or at one or more sites in the amino acidsequence, or insertion of one or more amino acids at one or more sitesin the amino acid sequence. For example, the nucleotide sequences of thepresent invention (SEQ ID NOs.: 1-6) are the T. reesei genomic sequencesthat are the borders of the insertional point of the pyr4 gene andrepresent the gene(s) that has been disrupted in the T. reesei genome.

As used herein, “percent (%) sequence identity” with respect to theamino acid or nucleotides sequences identified herein is defined as thepercentage of amino acid residues or nucleotides in a candidate sequencethat are identical with the amino acid residues or nucleotides in asequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Methods for performing sequence alignment and determiningsequence identity are known to the skilled artisan, may be performedwithout undue experimentation, and calculations of identity values maybe obtained with definiteness. See, for example, Ausubel, et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 19 (GreenePublishing and Wiley-Interscience, New York); and the ALIGN program(Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3(National Biomedical Research Foundation, Washington, D.C.). A number ofalgorithms are available for aligning sequences and determining sequenceidentity and include, for example, the homology alignment algorithm ofNeedleman, et al., (1970) J. Mol. Biol. 48:443; the local homologyalgorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482; the searchfor similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci.85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187(1997); and BLASTP, BLASTN, and BLASTX algorithms (see, Altschul, etal., (1990) J. Mol. Biol. 215:403-410). Computerized programs usingthese algorithms are also available, and include, but are not limitedto: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul, etal., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul,et al., supra, FASTA, and TFASTA, available in the Genetics ComputingGroup (GCG) package, Version 8, Madison, Wis., USA; and CLUSTAL in thePC/Gene program by Intelligenetics, Mountain View, Calif. Those skilledin the art can determine appropriate parameters for measuring alignment,including algorithms needed to achieve maximal alignment over the lengthof the sequences being compared. Preferably, the sequence identity isdetermined using the default parameters determined by the program.Specifically, sequence identity can be determined by the Smith-Watermanhomology search algorithm (Meth. Mol. Biol. 70:173-187 (1997)) asimplemented in MSPRCH program (Oxford Molecular) using an affine gapsearch with the following search parameters: gap open penalty of 12, andgap extension penalty of 1. Preferably, paired amino acid comparisonscan be carried out using the GAP program of the GCG sequence analysissoftware package of Genetics Computer Group, Inc., Madison, Wis.,employing the blosum62 amino acid substitution matrix, with a gap weightof 12 and a length weight of 2. With respect to optimal alignment of twoamino acid sequences, the contiguous segment of the variant amino acidsequence may have additional amino acid residues or deleted amino acidresidues with respect to the reference amino acid sequence. Thecontiguous segment used for comparison to the reference amino acidsequence will include at least 20 contiguous amino acid residues and maybe 30, 40, 50 or more amino acid residues. Corrections for increasedsequence identity associated with inclusion of gaps in the derivative'samino acid sequence can be made by assigning gap penalties.

The term “% homology” is used interchangeably herein with the term “%identity”

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,and are publicly available on the Internet (see, for example, the BLASTpage on the National Center for Biotechnology Information website). Seealso, Altschul, et al., 1990 and Altschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, et al., 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

As used herein, “expression vector” means a DNA construct including aDNA sequence which is operably linked to a suitable control sequencecapable of affecting the replication, disruption, or expression of theDNA in a suitable host. Such control sequences may include origins ofreplication or a promoter to affect transcription, an optional operatorsequence to control transcription, a sequence encoding suitableribosome-binding sites on the mRNA, and sequences which controltermination of transcription and translation. Different cell types arepreferably used with different expression vectors. A preferred promoterfor vectors used in Bacillus subtilis is the AprE promoter; a preferredpromoter used in E. coli is the Lac promoter, a preferred promoter usedin Saccharomyces cerevisiae is PGK1, a preferred promoter used inAspergillus niger is glaA, and preferred promoters for Trichodermareesei are reesei cbh1, cbh2, eg1, eg2, eg3, eg5, xln1 and xln2promoters. The vector may be a plasmid, a phage particle, or simply apotential genomic insert. Once transformed into a suitable host, thevector may replicate and function independently of the host genome, ormay, under suitable conditions, integrate into the genome itself. In thepresent specification, plasmid and vector are sometimes usedinterchangeably. However, the invention is intended to include otherforms of expression vectors which serve equivalent functions and whichare, or become, known in the art. Thus, a wide variety ofhost/expression vector combinations may be employed in expressing orreplicating the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences such as various knownderivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.coli including col E1, pCR1, pBR322, pMb9, pUC 19 and their derivatives,wider host range plasmids, e.g., RP4, phage DNAs e.g., the numerousderivatives of phage, e.g., NM989, and other DNA phages, e.g., M13 andfilamentous single stranded DNA phages, yeast plasmids such as the 2μplasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in animal cells and vectors derived from combinationsof plasmids and phage DNAs, such as plasmids which have been modified toemploy phage DNA or other expression control sequences. Expressiontechniques using the expression vectors of the present invention areknown in the art and are described generally in, for example, Sambrook,et al., MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION, ColdSpring Harbor Press (1989). Often, such expression vectors including theDNA sequences of the invention are transformed into a unicellular hostby direct insertion into the genome of a particular species through anintegration event (see, e.g., Bennett & Lasure, MORE GENE MANIPULATIONSIN FUNGI, Academic Press, San Diego, pp. 70-76 (1991) and articles citedtherein describing targeted genomic insertion in fungal hosts).

“pPZP100,” as used herein, refers to 1) an expression vector that can betransformed into T. reesei, and also 2) a shuttle vector that can beamplified in E. coli and Agrobacterium. See, for example, Hajdukiewiezet al. (1994) Plant Mol. Bio. 25:989-994.

As used herein, “host strain” or “host cell” means a suitable host foran expression vector including DNA according to the present invention.Host cells useful in the present invention are generally prokaryotic oreukaryotic hosts, including any transformable microorganism in whichexpression can be achieved. Specifically, host strains may be Bacillussubtilis, Escherichia coli, Trichoderma reesei, Saccharomyces cerevisiaeor Aspergillus niger. Host cells are transformed or transfected withvectors constructed using recombinant DNA techniques.

A “modified cell” or “modified strain” means a cell or strain that hasbeen modified by having one of the nucleic acid sequences describedherein deleted or inactivated (e.g., disrupted).

An “inactivated gene” means locus of a genome that, prior to itsinactivation, was capable of producing a protein, i.e., capable of beingtranscribed into an RNA that can be translated to produce a full lengthpolypeptide. A gene is inactivated when it is not transcribed andtranslated into full length catalytically active protein. A gene may beinactivated by altering a sequence required for its transcription, byaltering a sequence required for RNA processing, e.g., poly-A tailaddition by altering a sequence required for translation, for example. Adeleted gene, a gene containing a deleted region, a gene containing arearranged region, a gene having an inactivating point mutation orframeshift and a gene containing an insertion are types of inactivatedgene. A gene may also be inactivated using antisense or any other methodthat abolishes expression of that gene.

As used herein, “functionally attached” or “operably linked” means thata regulatory region, such as a promoter, terminator, secretion signal orenhancer region is attached to or linked to a structural gene andcontrols the expression of that gene.

As used herein, a substance (e.g., a polynucleotide or protein) “derivedfrom” a microorganism means that the substance is native to themicroorganism.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota. The filamentous fungi are characterized byvegetative mycelium having a cell wall composed of chitin, glucan,chitosan, mannan, and other complex polysaccharides, with vegetativegrowth by hyphal elongation and carbon catabolism that is obligatelyaerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Trichoderma, e.g., Trichodermareesei, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum;Penicillium sp.; Humicola sp., including Humicola insolens;Chrysosporium sp., including C. lucknowense; Gliocladium sp.;Aspergillus sp.; Fusarium sp., Neurospora sp., Hypocrea sp., andEmericella sp. As used herein, the term “Trichoderma” or “Trichodermasp.” refers to any fungal strains which have previously been classifiedas Trichoderma or are currently classified as Trichoderma.

In one preferred embodiment, the filamentous fungal parent cell is anAspergillus niger, Aspergillus awamori, Aspergillus aculeatus, orAspergillus nidulans cell.

In another preferred embodiment, the filamentous fungal parent cell is aTrichoderma reesei cell.

“Trichoderma” or “Trichoderma sp.” refers to any fungal strains whichhave previously been classified as Trichoderma or which are currentlyclassified as Trichoderma. Preferably the species are Trichoderma reeseior Trichoderma viride.

The term “equivalent” refers to nucleotide sequences encodingfunctionally equivalent polypeptides or functionally equivalentpolypeptides; Equivalent nucleotide sequences will include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants, and include sequences that differfrom a native or natural nucleotide due to the degeneracy of the geneticcode.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Mullis, et al., U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins, eds., 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins, eds., 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H.Miller and M. P. Calos, eds., 1987, Cold Spring Harbor Laboratory);Methods In Enzymology, Vols. 154 and 155 (Wu, et al., eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986)). Also, information regarding methods ofpreparation, expression, isolation and use of proteases may be obtainedby review of U.S. Pat. No. 6,768,001. Terms not defined within thisdocument either specifically, by reference or by context are to havedefinitions common in the art at the time of filing.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

The invention will now be described in detail by way of reference onlyusing the following definitions and examples. All patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively. Practitioners are particularly directed toSambrook, et al., 1989, and Ausubel, F M, et al., 1993, for definitionsand terms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

The applicants have identified the disrupted nucleotide sequencesresponsible for the increased production of proteins, including but notlimited to cellulose. The nucleotide sequences are referred to as 7p,8k, 7E, 9G, 8Q and 203 from T. reesei, which are presented in FIG. 1,and are SEQ ID NOS.: 1-6, respectively Likewise, it is contemplated thatthe nucleotide sequences of the present invention are used in screeningassays for the detection of, e.g., other strains of T. reesei or othermicroorganisms effective in protein production or for homologs andsequence variants of the sequences of the present invention.

It is a preferred embodiment of the present invention that the novel T.reesei strains of the present invention are used in methods for theproduction of proteins wherein the proteins may be heterologous orhomologous to the host cell. For example, a method is contemplatedwherein a suitable sterile growth medium is inoculated with one or morestrains of T. reesei selected from the group consisting of T. reeseistrains that have had at least one nucleotide sequence selected from 7p,8k, 7E, 9G, 8Q and 203 deleted or inactivated and the inoculated growthmedium is incubated under conditions which will permit the growth ofsaid T. reesei strain. The present invention is not limited to anyparticular growth/culture medium. Any complex or defined medium thatsupports growth and is conductive of protein production and inparticular cellulase production is suitable. Examples include mediadisclosed in WO 2005/118795 or media disclosed in Ilmen, M., Saloheimo,A., Onnela, M., and Penttila, M. E., 1997, App Environ Microbiol 63,1298-1306. In a preferred embodiment, 100 mM PIPPS (Calbiochem) wasincluded to maintain the pH at 5.5. Also, the present invention is notlimited to any particular culture method (e.g., batch culture,continuous flow culture, etc.). It is further contemplated that thesterile growth medium may additionally comprise an inducer of cellulaseproduction. Non-limiting examples of suitable inducers are cellulose,lactose, sophorose and glucose/sophorose. In an embodiment the inducerof cellulase production is glucose/sophorose as described in USPublication Number US-2004-0121446.

It is also an embodiment of the present invention that the cellulases(in the form of “whole cellulase”) produced by the T. reesei strains ofthe present invention are purified from the culture medium.

Reverse Genetics

One of the objectives of much of genetic research is to identify thegenes responsible for selected phenotypic traits. While much effort isbeing undertaken to develop genomic information from a large number oforganisms, often the information about the function of a gene is moreimportant than the information as to the sequence of the gene itself.One way in which the function of individual genes is studied is to lookfor mutated versions of the gene of interest. Sometimes the search formutated versions of a gene and the study of the mutated genes isreferred to as “reverse genetics.” If one finds a gene which is mutatedso as to render the mutated gene inoperative, one can discern whatphenotypic change has been make to the organism that renders itdifferent from organisms not carrying the mutated version of the gene.

Various strategies have been developed for using reverse genetics tostudy the functioning of genes. For example, one laboratory at theUniversity of Wisconsin has created a large population of Arabidopsisplant lines each of which had been transformed using the transferred-DNA(T-DNA) from the bacteria Agrobacterium tumefaciens, which has thenative ability to transfer T-DNA into the genome of the plant cell.

In the present invention, a large population of T. reesei fungal lineshas been created to screen for mutant strains with increased cellulaseproduction. The techniques used were based on work by Sessions, et al.,(A high-throughput Arabidopsis reverse genetics system, The Plant Cell,14:2985-2994 (2002)). In the present invention, the pyr4 gene wastransfected into T. reesei using Agrobacterium T-DNA border sequences tocause disruptions of the genomic DNA. The transformants were thenscreened for strains that showed increased cellulase production ascompared to the parent T. reesei strain and as exemplified below.

Molecular Biology

The techniques of molecular biology are used in the present inventionfor the purpose of identifying and isolating mutant strains of T. reeseithat have increased effectiveness in the production of cellulases overthe parent strain.

In one embodiment this invention provides for the identification ofgenes and gene mutations of Trichoderma reesei that confer an increasein cellulase productivity to T. reesei. Therefore, this invention relieson routine techniques in the field of recombinant genetics and reversegenetics (discussed above and in the Examples section). Basic textsdisclosing the general methods of use in this invention includeSambrook, et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andAusubel, et al., eds., Current Protocols in Molecular Biology (1994).

In one embodiment of the present invention, disruption libraries of T.reesei were prepared by transforming in the pyr4 gene usingAgrobacterium tumefaciens-mediated transformation. In anotherembodiment, Agrobacterium rhizogenes is used for the transformationprotocols. In yet another embodiment, the bacterium used for thetransformation protocol is any species of Agrobacterium (Agrobacteriumsp.) suitable for the purpose. Other bacterial strains useful forinsertional mutagenesis are known to those skilled in the art. See forexample, Constans, A. (2005) The Scientist 19(5):32; Broothaerts et al.(2005) Nature 433:629-633; and Gelvin, S B (2005) Nature 433:583-584.FIG. 2 exemplifies a suitable expression vector. In one embodiment, theexpression vector used serves a dual function in that it is capable ofbeing replicated in E. coli and in Agrobacterium using ColE1 and pVS1plasmid origins for replication, respectively. The expression vectorused in the present invention was pPZP100 but one practiced in the artwill understand that other vectors and other plasmid origins ofreplication are known in the art and are also effective for thesepurposes. Those skilled in the art are also aware that a natural plasmidorigins of replication can be modified by replacement, substitution,addition or elimination of one or more nucleotides without changing itsfunction or can be replaced with other effective plasmid origins ofreplication. The practice of the invention encompasses and is notconstrained by such alterations to or replacement of the plasmid originsof replication or by the use of other plasmids, origins of replicationor transfection methods known in the art at the time of this inventionor by their equivalents.

The expression vector/construct typically contains a transcription unitor expression cassette that contains all the additional elementsrequired for the expression of the heterologous sequence. A typicalexpression cassette thus contains a promoter operably linked to theheterologous nucleic acid sequence and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

The practice of the invention is not constrained by the choice ofpromoter in the genetic construct. However, exemplary promoters are theTrichoderma reesei cbh1, cbh2, eg1, eg2, eg3, eg5, xln1 and xln2promoters.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

Although any fungal terminator is likely to be functional in the presentinvention, preferred terminators include: the terminator fromAspergillus nidulans trpC gene (Yelton, M., et al., (1984) PNAS USA81:1470-1474, Mullaney, E. J., et al., (1985) MGG 199:37-45), theAspergillus awamori or Aspergillus niger glucoamylase genes (Nunberg, J.H., et al., (1984) Mol. Cell. Biol. 4:2306, Boel, E., et al., (1984)EMBO J. 3:1581-1585) and the Mucor miehei carboxyl protease gene (EPOPublication No. 0 215 594).

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for Agrobacterium transformation expression inplants and other eukaryotic cells may be used. Suitable vectors include,but are not limited to, the pPZP family of Agrobacterium binary vectors(as described in Hjdukiewiez et al 1994. Plant Molecular biology 25:989-994), pCAMBIA 1300 (as described in Mullins, E. D. et al 2001.Phytopathology 91:173-180), pUR5755 (as described in Gouka, R. K. et al1999. Nature biotechnology 17: 598-601) and M13, as well as plasmidssuch as pBR322 based plasmids, pSKF, pET23D, and fusion expressionsystems such as MBP, GST, and LacZ. Epitope tags can also be added torecombinant proteins to provide convenient methods of isolation, e.g.,c-myc.

The elements that are typically included in expression vectors alsoinclude a replicon, a gene encoding antibiotic resistance to permitselection of bacteria that harbor recombinant plasmids, and uniquerestriction sites in nonessential regions of the plasmid to allowinsertion of heterologous sequences. The particular antibioticresistance gene chosen is not critical, any of the many resistance genesknown in the art are suitable. The prokaryotic sequences are preferablychosen such that they do not interfere with the replication orintegration of the DNA in Trichoderma reesei. For Agrobacteriumtransformation it is necessary to use sequences that allow replicationin both E. coli and Agrobacterium as well as the left and right bordersof the Agrobacterium Ti plasmid. Non-limiting examples of suitablesequences are given in the Examples section, infra.

The methods of transformation of the present invention may result in thestable integration of all or part of the transformation vector into thegenome of the filamentous fungus. However, transformation resulting inthe maintenance of a self-replicating extra-chromosomal transformationvector is also contemplated.

Many standard transfection methods can be used to produce Trichodermareesei cell lines that express large quantities of the heterologousprotein. Some of the published methods for the introduction of DNAconstructs into cellulase-producing strains of Trichoderma includeLorito, Hayes, DiPietro and Harman, 1993, Curr. Genet. 24: 349-356;Goldman, VanMontagu and Herrera-Estrella, 1990, Curr. Genet. 17:169-174;Penttila, Nevalainen, Ratto, Salminen and Knowles, 1987, Gene 6:155-164, for Aspergillus Yelton, Hamer and Timberlake, 1984, Proc. Natl.Acad. Sci. USA 81: 1470-1474, for Fusarium Bajar, Podila andKolattukudy, 1991, Proc. Natl. Acad. Sci. USA 88: 8202-8212, forStreptomyces Hopwood, et al., 1985, The John Innes Foundation, Norwich,UK and for Bacillus Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi,1990, FEMS Microbiol. Lett. 55: 135-138.

However, any of the well-known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, biolistics, liposomes, microinjection, plasma vectors,viral vectors and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). Also ofuse is the Agrobacterium-mediated transfection method described in U.S.Pat. No. 6,255,115. It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing atleast one gene into the host cell capable of expressing the heterologousgene.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofgenes under control of protease gene promoter sequences. Large batchesof transformed cells can be cultured as described in Example 2, infra.Finally, product is recovered from the culture using standardtechniques.

Thus, the invention herein provides for the expression and enhancedsecretion of desired polypeptides whose expression is under control ofgene promoter sequences including naturally occurring protease orcellulase genes, fusion DNA sequences, and various heterologousconstructs. The invention also provides processes for expressing andsecreting high levels of such desired polypeptides.

Host Cells

In certain embodiments, the cell is a filamentous fugal cell having agenome comprising an inactivated gene, where the inactivated gene thatcomprises a nucleotide sequence that is least 95% identical to any ofSEQ ID NOs:1-6.

Genes may be inactivated in a fungal cell using a number of methods,including methods that employ antisense molecules, RNA interference, orribozymes, for example. In certain embodiments, however, expression ofthe genes may be reduced by gene inactivation.

A subject fungal cell may be constructed using any convenient method,for example, by altering the sequence of a gene of the cell by making aninsertion, deletion, replacement, or rearrangement in the gene forexample. The portion of the gene to be altered may be, for example, thecoding region or a regulatory element required for expression of thecoding region. An example of such a regulatory or control sequence of agene may be a promoter sequence or a functional part thereof, i.e., apart which is necessary for expression of the gene.

In one embodiment, the subject fungal cell may be constructed by genedeletion methods. Gene deletion techniques enable the partial orcomplete removal of the gene thereby eliminating their expression. Insuch methods, the deletion of the gene may be accomplished by homologousrecombination using a plasmid that has been constructed to contiguouslycontain the 5′ and 3′ regions flanking the gene.

In another embodiment, the subject fungal cell may be constructed byintroducing, substituting, and/or removing one or more nucleotides inthe gene or a regulatory element thereof required for the transcriptionor translation thereof. For example, nucleotides may be inserted orremoved so as to result in the introduction of a stop codon, the removalof the start codon, removal of a splice cite, or a frame-shift of theopen reading frame. Such a modification may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. See, for example, Botstein and Shortie,1985, Science 229: 4719; Lo et al., 1985, Proceedings of the NationalAcademy of Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic AcidsResearch 16: 7351; Shimada, 1996, Meth. Mol. Biol. 57: 157; Ho et al.,1989, Gene 77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar andSommer, 1990, BioTechniques 8: 404.

In another embodiment, the subject fungal cell may be constructed bygene disruption techniques by inserting into the gene of interest anintegrative plasmid containing a nucleic acid fragment homologous to thegene which will create a duplication of the region of homology andincorporate vector DNA between the duplicated regions. Such genedisruption can eliminate gene expression if the inserted vectorseparates the promoter of the gene from the coding region or interruptsthe coding sequence such that a non-functional gene product results. Adisrupting construct may be simply a selectable marker gene accompaniedby 5′ and 3′ regions homologous to the gene. The selectable markerenables identification of transformants containing the disrupted gene.

In another embodiment, the subject fungal cell may be constructed by theprocess of gene conversion (see, for example, Iglesias and Trautner,1983, Molecular General Genetics 189: 73-76). For example, in the geneconversion method, a nucleotide sequence corresponding to the gene(s) ismutagenized in vitro to produce a defective nucleotide sequence which isthen transformed into the parent strain to produce a defective gene. Byhomologous recombination, the defective nucleotide sequence replaces theendogenous gene.

In an alternative embodiment, the subject fungal cell may be constructedusing random or specific mutagenesis using methods that include, but arenot limited to, chemical mutagenesis (see, for example, Hopwood, TheIsolation of Mutants in Methods in Microbiology (J. R. Norris and D. W.Ribbons, eds.) pp 363-433, Academic Press, New York, 1970) andinsertional mutagenesis, such as transposition (see, for example,Youngman et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2305-2309).Modification of the gene may be performed by subjecting the parentstrain to mutagenesis and screening for mutant strains in whichexpression of the gene has been reduced or eliminated. The mutagenesis,which may be specific or random, may be performed, for example, by useof a suitable physical or chemical mutagenizing agent, for example.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the parent strain to be mutagenized inthe presence of the mutagenizing agent of choice under suitableconditions, and selecting for mutants exhibiting reduced or noexpression of a gene.

As noted above, the subject fungal cell may be a filamentous fungalcell. In certain embodiments, the cell may be non-pathogenic, i.e.,non-pathogenic to humans. In particular embodiments, the cells may befilamentous fungal cells of a strain that has a history of use forproduction of proteins that has GRAS status, i.e., a GenerallyRecognized as Safe, by the FDA.

In particular embodiments, the subject fungal cell may be a cell of thefollowing species: Trichoderma, (e.g., Trichoderma reesei (previouslyclassified as T. longibrachiatum and currently also known as Hypocreajecorina), Trichoderma viride, Trichoderma koningii, and Trichodermaharzianum)); Penicillium sp., Humicola sp. (e.g., Humicola insolens andHumicola grisea); Chrysosporium sp. (e.g., C. lucknowense), Gliocladiumsp., Aspergillus sp. (e.g., Aspergillus oryzae, Aspergillus niger,Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus,Aspergillus japonicus, Aspergillus sojae, and Aspergillus awamori),Fusarium sp., Neurospora sp., Hypocrea sp., or Emericella sp. (See also,Innis et al., (1985) Sci. 228:21-26), among others. In some embodiments,subject fungal cells may be strains of Aspergillus niger which includeATCC 22342, ATCC 44733, ATCC 14331 and strains derived therefrom. Insome embodiments, a host cell may be one wherein native genes have beendeleted or inactivated. For example genes corresponding to proteasegenes or genes corresponding to cellulase genes may be inactivated.

In one embodiment, the subject fungal cell may contain a recombinantnucleic acid for expression of a protein in the cell. The protein may benot native to the cell (i.e., heterologous) or native to the cell (i.e.,endogenous to the cell). The protein may be expressed using a number ofdifferent protocols, e.g., by use of an expression cassette forproduction of the protein, by operably linking a nucleic acid encodingthe protein to a promoter that is part of the genome of the cell withanother promoter, or by replacing the promoter that is part of thegenome of the cell, for example.

The DNA sequences of several fungal genes and the proteins encoded bythose genes have been determined and deposited into NCBI's Genbankdatabase, including the complete genomes of Aspergillus fumigatus,Candida glabrata, Cryptococcus neoformans, Debaryomyces hansenii,Encephalitozoon cuniculi, Eremothecium gossypii, Gibberella zeae,Kluyveromyces lactis, Magnaporthe grisea, Neurospora crassa, Pichiastipitis, Saccharomyces cerevisiae (baker's yeast), Schizosaccharomycespombe (fission yeast), Ustilago maydis and Yarrowia lipolytica. Furthersequences may be found at the US Department of Energy Joint GenomeInstitute's Trichoderma reesei and Aspergillus niger genome sequencedatabases (as found at the world wide website of jgi.doe.gov).

In certain embodiments, a subject gene may comprise a nucleotidesequence that is at least 70% (e.g., at least 80%, at least 90%, atleast 95%, at least 97% or at least 98% sequence identity) to any of SEQID NOS:1-6; or b) may hybridize under stringent conditions to any of SEQID NOS:1-6.

Protein of Interest or Desired Protein

The terms protein of interest and desired protein may be usedinterchangeably herein. The present invention is particularly useful inenhancing the intracellular and/or extracellular production of proteins.The protein may be homologous or heterologous. Proteins that mayproduced by the instant invention include, but are not limited to,hormones, enzymes, growth factors, cytokines, antibodies and the like.

Hormones include, but are not limited to, follicle-stimulating hormone,luteinizing hormone, corticotropin-releasing factor, somatostatin,gonadotropin hormone, vasopressin, oxytocin, erythropoietin, insulin andthe like.

Growth factors are proteins that bind to receptors on the cell surface,with the primary result of activating cellular proliferation and/ordifferentiation. Growth factors include, but are not limited to,platelet-derived growth factor, epidermal growth factor, nerve growthfactor, fibroblast growth factors, insulin-like growth factors,transforming growth factors and the like.

Cytokines are a unique family of growth factors. Secreted primarily fromleukocytes, cytokines stimulate both the humoral and cellular immuneresponses, as well as the activation of phagocytic cells. Cytokinesinclude, but are not limited to, colony stimulating factors, theinterleukins (IL-1 (α and β), IL-2 through IL-13) and the interferons(α, β and γ).

Human Interleukin-3 (IL-3) is a 15 kDa protein containing 133 amino acidresidues. IL-3 is a species specific colony stimulating factor whichstimulates colony formation of megakaryocytes, neutrophils, andmacrophages from bone marrow cultures.

Antibodies include, but are not limited to, immunoglobulins from anyspecies from which it is desirable to produce large quantities. It isespecially preferred that the antibodies are human antibodies.Immunoglobulins may be from any class, i.e., G, A, M, E or D.

Additionally, a “protein of interest” or “polypeptide of interest”refers to the protein to be expressed and secreted by the host cell. Theprotein of interest may be any protein that up until now has beenconsidered for expression in prokaryotes. In one embodiment, the proteinof interest which is expressed and secreted include proteins comprisinga signal peptide. The protein of interest may be either homologous orheterologous to the host. Thus, a protein of interest may be a secretedpolypeptide particularly an enzyme which is selected from amylolyticenzymes, proteolytic enzymes, cellulolytic enzymes, oxidoreductaseenzymes and plant wall degrading enzymes. Examples of these enzymesinclude amylases, proteases, xylanases, lipases, laccases, phenoloxidases, oxidases, cutinases, cellulases, hemicellulases, esterases,perioxidases, catalases, glucose oxidases, phytases, pectinases,glucosidases, isomerases, transferases, galactosidases and chitinases.The secreted polypeptide may also be a hormone, a growth factor, areceptor, vaccine, antibody or the like. In an embodiment the secretedpolypeptide is a cellulolytic enzyme.

Industrial Applications of the Invention

The present invention has many practical applications in industry, as iscontemplated herein, this description is intended to be exemplary, andnon-inclusive. The T. reesei strains of the present invention are moreeffective in cellulase production over the parent strain and, as such,are useful in the efficient production of cellulases that are useful invarious industries as exemplified below.

In several embodiments, cellulase produced by the T. reesei strains ofthe present invention have contemplated use in ethanol production,baking, fruit juice production, brewing, distilling, wine making,leather, oils and fats, paper and pulp and the animal feed production.

In other embodiments, the present invention has contemplated is theactive “biological” component of detergents and cleaning products. Here,cellulases are used to break down various stains and other acquiredcontaminants Embodiments of the invention include testing thecompatibility of enzymes with detergent ingredients by doing stabilitystudies and testing them in a variety of formulations.

In another embodiment, the cellulases produced by the T. reesei strainsof the present invention have contemplated use in the textile industry,mainly in the finishing of fabrics and garments. Major applicationsinclude: desizing, removal of size, (that is, removal of stiff elementsof fiber), from threads in fabrics after weaving. For example, thecellulases produced by the present invention can be used inbio-polishing, a process to reduce or eliminate pilling tendency and togive fabrics a smoother and glossier appearance, and in bio-stoning, aprocess that can replace traditional pumice stones used in stonewashingof denim to achieve a worn look.

In yet another embodiment, the present invention has contemplatedenzymatic uses for the liquefaction and saccharification of starch intoglucose and isomerisation into fructose. The cellulases produced by thepresent invention may be used to convert large volumes of corn and othergrains into sweeteners, like high fructose corn syrup and maltose syrup.

It will be apparent to those skilled in the art to which this inventionpertains that other embodiments of the present invention may beperformed based on the teachings contained herein. It is intended thatsuch embodiments are contemplated to be included within the scope of thepresent invention.

EXAMPLES

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed in any way. The attached Figures are meant to beconsidered as integral parts of the specification and description of theinvention. All references cited are herein specifically incorporated byreference for all that is described therein. The following examples areoffered to illustrate, but not to limit the claimed invention.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); U (units); M (Molar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); pmole (pico moles); g (grams); mg(milligrams); kg (kilograms); μg (micrograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); h (hours);min (minutes); sec (seconds); msec (milliseconds), MM (Minimal Medium).

Example 1 Agrobacterium-Mediated Transformation Procedures

This Example shows how insertional mutagenesis was performed usingAgrobacterium tumefaciens-mediated transformation. Likewise,Agrobacterium rhizogenes is equally effective for use in thetransformation procedure. This procedure allowed the production ofmutant libraries of Trichoderma reesei that contained strains withsingle disruption events in the genomic DNA. These disruption eventswere randomly distributed throughout the genome. Since the disruptionwas done using a known DNA sequence, it was possible to trace the exactsite of disruption and identify the gene(s) that were affected. In thiscase, the libraries were screened for improved cellulase producers (see,Example 2). In strains 7p, 8k, 7E, 9G, 8Q and 203, the specificdisruptive genomic DNA sequences responsible for the improvement incellulase production were identified.

Disruption libraries of Trichoderma reesei were prepared by transformingin the pyr4 gene using Agrobacterium tumefaciens-mediatedtransformation. The disruption library contained about 30,000transformants. The disruption library was screened using the Toyamamethod (see, Example 2). This method selects for mutant that are able toutilize and/or grow more efficiently on Avicel (cellulose). In the past,this method has resulted in the isolation of strains with improved yieldand productivity. Mutants isolated from the Toyama screen were examinedin shake flasks for total protein production. Mutants showing increasedprotein production of greater that 10% in multiple experiments wereconsidered to be improved. One mutant, 8k, was found to have improvedtotal protein production compared to the parental control. Southernanalysis showed that this strain contained only one copy of the pyr4gene indicating that one disruption event had taken place. The sequenceof the T. reesei sequence in 8k that was disrupted by the pyr4 gene wasdetermined by thermal asymmetric interlace (tail) PCR. BLAST resultswere obtained from public databases as well as the T. reesei genomedatabase. For 8k, the disrupted sequence immediately after the leftborder matched bases 1263481-1253276 in scaffold-4 in the T. reeseigenome database. For 7p, the disrupted sequence immediately after theleft border matched bases 229978-229764 in scaffold-4 when BLASTed inthe T. reesei genome library. The sequence for the other four strains ofthe present invention were identified similarly.

The Agrobacterium tumefaciens strain used for this work was strain EHA105. EHA 105 is considered to be a hypervirulent strain (Hood, et al.,1993). Other strains are also compatible with the following procedureare, e.g., A136 and EHA 101. Transformation frequencies for these threestrains are similar when transforming T. reesei. In addition, A.rhizogenes (ATCC 43057) or any other rhizogenes strain may be usedherein.

The PZP 100-based expression vector was made as follows. The vectorcontains the left and right T-DNA border regions, a pBR322 bam site formobilization from E. coli to Agrobacterium, CalE1 and pVS1 plasmidorigins for replication in E. coli and Agrobacterium, respectively.Bacterial markers confer resistance to amp (Hajdukiewiez, O., et al.,1994). A representation of the vector in shown in FIG. 2.

The E. coli vector was made as follows. The expression cassette wasprepared by standard molecular biological techniques and ligated into aPZP vector. Preferred strains of E. coli are XL gold cells (Invitrogen,Carlsbad, Calif.) and DH5α, which is known in the art. LA plus 25 ppmcmp plates were used to select for E. coli transformants. Typically,about 1-10% of the E. coli transformants have the desired vector. LBplus 25 ppm cmp was used to grow the E. coli containing the vector DNA.Vector DNA was isolated using standard protocols known in the art.

Vector DNA was electroporated into Agrobacterium cells as follows.First, competent Agrobacterium cells were prepared. Agrobacterium cellswere revived from cryopreservation by growing on LA medium at 28° C. forabout three days. Colonies were selected and grown in Luria-Bertaniculture broth (LB; Invitrogen) plus 0.1% glucose in 250 ml dented bottomflasks containing 50 ml medium. The cultures were incubated at 28° C.until growth occurred (about two days). An alternate procedure is tostart the culture in a 5 ml culture tube and transfer to the 250 mlflask when growth is noticed. About 10% of the volume (v/v) of the aboveflask was then transferred into a fresh flask with the same medium. Thisflask was incubated until an O.D. (at 600 nm) of about 0.4-0.8 wasobtained (about 5-6 hours of growth). Next, in the cold, the cells werespun down in a centrifuge at 10,000 rpm for 10 minutes. The cells werethen washed 3× in cold 1 M HEPES, pH 7.0. Next, the cells were washedonce in cold 1 mM HEPES with 10% glycerol. Aliquots of 50-100 ml werefroze at −70° C. Cell viability was determined (typically about 1×10⁹CFU/ml after freezing). Competent cells are good for one year or longerwhen stored at −70° C.

After the generation of competent Agrobacterium cells, the cells weretransfected by electroporation. Competent Agrobacterium calls werethawed on ice. About 40 μl of the cells were mixed with about 1 μg ofDNA in a 0.2 cm electroporation cell (on ice). The cells wereelectroporated at 200 Omnhs, at 25 μF, 2.5 volts with a Buchler 3-150electroporator. SOC medium (Invitrogen) was added immediately afterelectroporation into the electroporation tube. (In another embodiment,the Agrobacterium cells are electroporated with the ligation mixturethus skipping the E. coli step. With this alternate method, 1 μl of theligation mixture is used in the electroporation step.) After theaddition of SOC to the electroporation mixture, dilutions of the mixturewere plated onto LA medium plus 250 ppm cmp culture plates and incubatedat 28° C. for four days. (In other embodiment, as little at 25 ppm cmpcan be used to obtain colonies in a shorter time frame but a largernumber of colonies will need to be screened to find ones containing thevector. This is because some Agrobacterium strains have some naturalresistance to cmp). After electroporation, 1×10⁷ CFU/ml of Agrobacteriumtransformants were obtained and about 90-100% had the vector asdetermined by PCR.

Agrobacterium tumefaciens EHA 105 (Hajdukiewiez, P., Svab, Z., andMaliga, P. (1994) The small versatile, pPZP family of Agrobacteriumbinary vectors for plant transformation. Plant Molecular Biology 25,989-994) containing the PZP-pyr4 disruption vector was grown in 50 mL MMmedium at 28° C., 200 rpm, for 24 hr. After the 24 hr incubation a 5 mLaliquot was transferred to 50 mL Induction medium and incubated at 28°C., 200 rpm, until the absorbance at 600, reached 0.8 abs. About 10 mlof this culture was added to 20 ml fresh induction medium along with 10ml of a P-37 py4⁻ strain that had been grown for 24 hr at 28° C., 200rpm in YEG (g per liter: yeast extract—5, glucose—20, uridine—2 mg). Theviable count of this culture was about 1×10⁶ CFU/ml. The mixture wasincubated statically at 28° C., and sampled at 48, 72, 96, and 144 hr.Samples were washed 3× in sterile water, and plated on Vogels. Coloniesthat grew were transferred to a second Vogel plate for confirmation.Results are shown in the table below:

TABLE 1 Incubation # transformants time (h) isolated on vogels 0 0 48 272 5 96 7 144 4

These results indicate that Agrobacterium transformation can be done ina liquid medium when the incubation period is between 48-144 h. Theoptimal incubation time is between 72-96 h.

TABLE 2 Agrobacterium Induction Medium Make to one liter: K₃HPO₄ 2.05 gKH₂PO₄ 1.45 g NaCl 0.15 g MgSO₄*7H₂O 0.5 g CaCl₂*6H₂O 0.1 g FeSO₄*7H₂O0.0025 g (NH₄)₂SO₄ 0.5 g Glucose 1.8 g Glycerol 5.0 g Prepare in 40 mMMES buffer (2-(N-Morpholino)ethanesulfonic acid) pH 5.3 Aftersterilization add 1M acetosyringone 200 ml Agrobacterium Inductive PlateMedium Make to one liter: K₃HPO₄ 2.05 g KH₂PO₄ 1.45 g NaCl 0.15 gMgSO₄*7H₂O 0.5 g CaCl₂*6H₂O 0.1 g FeSO₄*7H₂O 0.0025 g (NH4)₂SO₄ 0.5 gGlucose 1.8 g Glycerol 5.0 g Agar 15 g Prepare in 40 mM MES pH 5.3 Aftersterilization add 1M acetosyringone 200 ml 100 mg/ml uridine 2.5 mlCool. Agrobacterium Minimal Medium (MM) Make to one liter: K₃HPO₄ 2.05 gKH₂PO₄ 1.45 g NaCl 0.15 g MgSO₄*7H₂O 0.5 g CaCl₂*6H₂O 0.1 g FeSO₄*7H₂O0.0025 g (NH₄)₂SO₄ 0.5 g Glucose 1.8 g After sterilization add:Chloramphenicol 25 U

Example 2 Tail-PCR

After screening T. reesei strains for improved cellulase production,improved strains were analyzed for genetic changes using mTAIL-PCR(modified thermal asymmetric interlaced-polymerase chain reaction).TAIL-PCR was performed using protocols modified from Sessions, et al.(The Plant Cell, 14:2985-2994, 2002) and Liu, et al. (Plant J.,8:457-463, 1995) and Liu and Whittier (Genomics, 25:674-661, 1995) usingprimers to the PZP vector and the gene of interest. Additionally,Southern blotting was performed to demonstrate that each of the newclones has only one copy of the modified gene.

Briefly, pyr4 specific primer and a pool of four arbitrary degenerate(AD) primers were used per round of TAIL-PCR cycling. The T-DNA primersused were as follows:

Random TAIL-PCR primers that were used:

Name: ad1 Synthesis: 50 nmole Purification: Salt-Free Sequence:NGTCGASWGANAWGAA [SEQ ID NO.: 7] Name: ad2 Synthesis: 50 nmolePurification: Salt-free Sequence: TGWGNAGSANCASAGA [SEQ ID NO.: 8] Name:ad3 Synthesis: 50 nmole Purification: Salt-Free Sequence:AGWGNAGWANCAWAGG [SEQ ID NO.: 9] Name: ad4 Synthesis: 50 nmolePurification: Salt-Free Sequence: WGTGNAGWANCANAGA [SEQ ID NO.: 10]

The final concentrations of the pooled primers were AD1 3.0 μM, AD2 3.0μM, AD3 3.0 μM and AD4 4.0 μM.

Specific Pyr4 Primer

Reverse

4rip [SEQ ID NO.: 11] 5′AGCCGCGGCCTCCTGAT-′3 5rip [SEQ ID NO.: 12]5′GTCGGCGCTCAGGCACAGGTTGG-′3 6rip [SEQ ID NO.: 13]5′CGTCGCCGTCTCGCTCCTG-′3

Nested Primer

Reverse

8rip [SEQ ID NO.: 14] 5′TGCGGGAGGAAGAGGAGTAGGAAC′3The tail-PCR procedure was performed as follows. (see, Sessions, et.al., 2002, The Plant Cell, vol. 14. pp. 2985-2994).

In hotstart tubes using pipette tips with cotton plugs:

Step 1.

Distilled H20 34 uL  10X buffer 5 uL 10 mM dNTP 2 uL specific pyr4primer 1 uL (50 pmole) ad1 primer 2 uL ad2 primer 2 uL ad3 primer 2 uLad4 primer 2 uLHeat 95, 90 seconds, cool to 4° C.

Step 2.

Distilled H20 43 uL  10X buffer 5 uL genomic DNA 1 uL (dilute our DNA ⅕with H₂0) Hercules polymerase 1 uLRun the first PCR Program, round 1 (T1). Cycling parameters are givenbelow.

Step. 3

Distilled H20 34 uL  10X buffer 5 uL 10 mM dNTP 2 uL specific nestedpyr4 primer 1 uL (50 pmole) ad1 primer 2 uL ad2 primer 2 uL ad3 primer 2uL ad4 primer 2 uLHeat 95, 90 seconds, cool to 4° C.

Step 4.

Distilled H20 39 uL  10X buffer 5 uL DNA template 5 uL (from step 2,after running T1) Hercules polymerase 1 uLRun a second PCR round (T2). Cycling parameters are given below.

Two rounds of mTAIL-PCR cycling were performed. Cycling parameters forthe first round (T1) were (1) 94° C. for 2 min and 95° C. for 1 min; (2)5 cycles of 94° C. for 30 s, 62° C. for 1 min, and 72° C. for 2.5 min,(3) 2 cycles of 94° C. for 30 s, 25° C. for 3 min (50% ramp), and 72° C.for 2.5 min (32% ramp); (4) 15 cycles of 94° C. for 10 s, 68° C. for 1min, 72° C. for 2.5 min, 94° C. for 10 s, 68° C. for 1 min, 72° C. for2.5 min, 94° C. for 10 s, 44° C. for 1 min and 72° C. for 2.5 min; and(5) 72° C. for 7 min.

Cycling parameters for the second round (T2) containing 8 rip as thenesting primer as well as the Ad pool of primers was as follows: (1) 94°C. for 3 min, (2) 5 cycles of 94° C. for 10 s, 64° C. for 1 min, and 72°C. for 2.5 min, (3) 15 cycles of 94° C. for 10 s, 64° C. for 1 min, and72° C. for 2.5 min, 94° C. for 10 s, 64° C. for 1 min, 72° C. for 2.5min, 94° C. for 10 s, 44° C. for 1 min, and 72° C. for 2.5 min, (4) 5cycles of 94° C. for 10 s, 44° C. for 1 min, and 72° C. for 3 min; and(5) 72° C. for 7 min. Tail-PCR products were purified and sequenced bytreatment with exonuclease 1 (2.5 U; Amersham) and shrimp alkalinephosphatase (0.5 U; Amersham; Piscataway, N.J.) for 20 min at 37° C.followed by 15 min at 80° C. Sequencing reactions were performed in a384-well format using the 8rip primer and one-eighth of the suggestedamount of BigDye terminators (Applied Biosystems; Foster City, Calif.)and run on a standard sequencer. Sequencing reactions were passedthrough a Sephadex G-50 matrix to remove salts and unincorporated dueterminators. Resulting sequences were BLASTED against T. reesei genomicsequences at JGI Trichoderma reesei v.1.0.

Example 3 Fungal Transformation Procedures

Agrobacterium inoculate was prepared as follows. Twenty-five ml ofMinimal Medium (MM) in 250 ml flasks was inoculated with either a frozenstock of vector transformed Agrobacterium or inoculated directly from afresh LA plate culture. The culture was then incubated at 28° C. withshaking until the culture became cloudy (overnight to several daystime). Next, 10 ml was transferred to 50 ml of Induction Medium (IM) in250 ml flasks. Staring OD was about 0.1 at 600 nm and cells werecultured until OD was between 0.4-0.8. A fresh fungal plate (e.g., T.reesei) was prepared by resuspending spores in 10 ml of sterile water.

Transformation of fungus (e.g., T. reesei and Aspergillus niger) wasperformed as follows. About 100 μl of Agrobacterium whole broth (OD0.4-0.8 at 600 nm) was mixed with about 100 μl of fungal spores (10⁷sfu/ml) in a tube. One practiced in the art will realize that otherrations of Agrobacterium to fungal spores will also produce satisfactoryresults. Next, 0.1 to 1.0 ml of the mix was plated onto induction platescontaining nitrocellulose filters. Induction plates were supplementedwith nutrients required by the fungi as needed to correct any auxotrophypresent in the fungi. The plates were incubated at about 18-28° C. forabout 24-48 hours for T. reesei. For Aspergillus niger the cultures wasincubated at about 20-24° C. for about 20-24 hours. Next, the filterswere transferred to selective medium (Vogels medium for T. reesei andminimal medium for A. niger). The medium was supplemented with 250 ppmcarb to kill/inhibit Agrobacterium growth. The cultures were thenincubated at 28° C. until growth is evident on the filter. This takes aslong as one to two weeks. The transformants were transferred toselective medium when they were ready for further analysis.

Example 4 Toyama Screen

The Toyama screen was developed by Drs. Hideo and Nobuo Toyama fromMiniamikyashu University in Japan. Toyama, H. and Toyama, N., Successiveconstruction of cellulase hyperproducers of Trichoderma usinghyperpolyploids. Appl. Biochem. Biotechnol. Spring 84-86:419-429, 2000.The method describe therein was modified from the original procedure tobe more effective at improving Genencor's Trichoderma reesei cellulaseproduction strains and to be a high through-put screen. This screen hasbeen used successfully to isolate strains with improvements in bothyield and productivity. Here, we used the screen to isolate strains 7p,8k, 7E, 9G, 8Q and 203 from the parental strain. These strains showimproved cellulase production over the parental T. reesei strain.

Mutagenized spores were prepared using insertional mutagenesis (asdetailed in Example 2). An aliquot of the mutational libraries wasfrozen at −70° C. A viable spore count was determined and the remaininglibrary was divided so that each aliquot contained about 10⁶ spores.

The Toyama screen medium was prepared an cooled to 55° C. in a waterbath. In an 82 mm petri dish an aliquot was dispensed in a circle about½ way from the center of the plates and the edge. Next, 10 ml of Toyamascreening medium was carefully added to the culture plates and the platewas swirled so that the spores were dispersed in the middle of the platebut not dispersed al the way to the edges of the plate (see FIG. 3).Alternately, the spores may be spread out with a sterile loop before theaddition of the Toyama medium. The medium was let harden for 5-10minutes. Another 25 ml of medium was added to the plates and let harden.Next, another 10 ml of medium was added to the plates and let harden.The plates were incubated overnight

The next day growth of the spores was examined using a dissectingmicroscope. Plates were checked every four hours. Isolates werecollected as follows. Only the first 1-3 isolates that reached thesurface of the agar were collected. Any colonies that came up around theedges of the plate (cheaters) were ignored. Isolates were collectedusing a sterile razor blade under the microscope being careful not todig into the surface of the agar. The collected samples were placed ontoPDA (potato-dextrose-agar; Difco, Gaithersburg, Md.) plates andincubated at 28° C. Once grown, the isolates were evaluated on acidswollen cellulose plates or were put directly into shaker flasks.

TABLE 3 Toyama Screening Medium Make to one liter: 50X Vogels StockSolution 30 ml Avicel 0.5 g Agar 20 g 50X Vogels Stock Solution Usingthree glass containers separately add: Na₃Citrate*2H₂O 150 g MgSO₄*7H₂O10 g CaCl₂*2H₂O 5 g Dissolve in diH₂O to 300 ml KH₂PO₄ 250 g Dissolve inDiH₂O to 500 ml NH₄NO₃ 100 g Dissolve in diH₂O to 200 ml Allow allcomponents to clear in diH₂O. Combine all solutions and add with mixing:Vogels Trace Element Solution 5 ml Vogels Biotin Solution 2.5 ml VogelsBiotin Solution Make to one liter: d-biotin 0.1 g Dissolve in diH₂O to1.0 L Vogels Trace Element Solution Make to one liter: Citric Acid 50 gZnSO₄*7H₂O 50 g Fe(NH₄)₂SO₄*6H₂O 10 g CuSO₄*5H₂O 2.5 g MnSO4*4H₂O 0.5 gH₃BO₃ 0.5 g NaMoO₄*2H₂O 0.5 g

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A filamentous fungus having a mutation or deletion of part or all ofa gene having the sequence selected from at least one sequence set forthin any one of SEQ ID NOs:1-6, and said mutation or deletion results inthe enhanced production of a desired polypeptide compared to the parentfilamentous fungus.
 2. The filamentous fungus of claim 1, wherein saidfilamentous fungus is capable of expressing a heterologous protein. 3.The filamentous fungus of claim 1, wherein said heterologous protein isselected from the group consisting of hormones, enzymes, growth factors,and cytokines.
 4. The filamentous fungus of claim 3 wherein saidheterologous protein is an enzyme.
 5. The filamentous fungus of claim 4wherein said enzyme is selected from the group consisting of proteases,carbohydrases, lipases, isomerases, racemases, epimerases, tautomerases,mutases, transferases, kinases and phosphatases.
 6. A method for theproduction of a heterologous protein in a transformed filamentous fungushost cell according to claim 1, comprising the steps of: (a) obtaining afilamentous fungus host cell comprising a nucleic acid encoding saidheterologous protein wherein said host cell contains a mutation ordeletion in at least one nucleic acid sequence having the sequence setforth in any one of SEQ ID NOs:1-6, wherein said mutation or deletionresults in the enhanced production of the heterologous protein comparedto a parent filamentous fungus: and (b) growing said filamentous fungushost cell under conditions suitable for the expression of saidheterologous protein.
 7. The method of claim 6, wherein said genecomprises the nucleic acid sequence set forth in SEQ ID NO:1.
 8. Anisolated nucleotide sequence selected from a group consisting of SEQ IDNOs: 1-6.
 9. The isolated nucleotide sequence according to claim 8wherein said sequence has been modified.
 10. The isolated nucleotidesequence according to claim 9 wherein said modification is selected fromtruncation, deletion, mutation or other means of inactivation.
 11. Avector comprising at least one of the nucleotide sequences according toclaim
 9. 12. A host cell transformed with a vector according to claim11.
 13. (canceled)
 14. A method of producing a heterologous desiredpolypeptide said method comprising (a) obtaining a parental host cellstrain; (b) transforming said parental cell strain with a vectorencoding a desired polypeptide; (c) transforming said parental cellstrain with a vector according to claim 11 to produce a modified hostcell; (d) selecting modified host cells that produce said heterologousdesired polypeptide; and (e) culturing said modified host cell in asuitable growth medium for production of said heterologous desiredpolypeptide wherein steps (b) and (c) may be done in any order orsimultaneously.
 15. The method of claim 14, wherein said suitable growthmedium additionally comprises an inducer of cellulase production. 16.The method of claim 15, wherein said inducer of cellulase production isselected from one or more of cellulose, lactose, sophorose andglucose/sophorose.
 17. The method of claim 14, wherein the methodadditionally comprises the at least partial purification of cellulasesproduced by said culture.
 18. A method for producing a novel strain ofT. reesei using insertional mutagenesis wherein said novel strain of T.reesei has enhanced cellulase production as compared to the parentstrain of T. reesei, comprising: (a) preparing a population of competentAgrobacterium sp. cells by electroporating into competent Agrobacteriumsp. cells an expression vector comprising, in operable condition, theleft and right T-DNA boarder regions, pV51 plasmid origins forreplication in Agrobacterium sp. and bacterial markers to conferresistance to chloramphenicol to create a population comprisingtransformed Agrobacterium sp. cells; (b) selecting for Agrobacterium sp.from said population of step (a); (c) inoculating a culture of T. reeseispores with the Agrobacterium sp. transformants of step (b) to create aninduction culture; (d) culturing said induction culture of step (c) atabout 18° C. and for about 24 hours to create a population; (e)transferring samples of said population of transformed T. reesei of step(d) to selective medium and isolating colonies of T. reesei effective indegrading cellulose; and (f) comparing the effectiveness of cellulosedegradation between the T. reesei of the isolated colonies of step (e)and the non-transformed parent strain, wherein said T. reesei of theisolated colonies of step (e) are enhanced to in cellulose degradationwhen compared to the non-transformed parent strain.
 19. The method ofclaim 18, wherein said Agrobacterium sp. cells are selected fromAgrobacterium tumefaciens and Agrobacterium rhizogenes.